Differential molecular profiles and associated functionalities characterize connective tissue grafts obtained at different locations and depths in the human palate

The present study aimed to assess the molecular profiles of subepithelial connective tissue grafts (CTGs) obtained at different locations and depths in the human palate. Sixty-four CTGs belonging to anterior deep (AD), anterior superficial (AS), posterior deep (PD), and posterior superficial (PS) groups were subjected to RNA-Sequencing and their transcriptomes were analyzed computationally. Functional correlations characterizing the CTG groups were validated by cell biological experiments using primary human palatal fibroblasts (HPFs) extracted from the CTGs. A clearly more pronounced location-dependent than depth-dependent difference between the grafts, with a minimal number of genes (4) showing no dependence on the location, was revealed. Epithelial, endothelial, and monocytic cell migration was strongly (P < 0.001) potentiated by AD- and PS-HPFs. Moreover, significantly increased expression of genes encoding C-C and C-X-C motif chemokine ligands as well as significantly (P < 0.01) activated p38 signaling suggested immunomodulatory phenotype for AD- and PS-HPFs. Increased growth factor gene expression and significantly activated (P < 0.001) Erk and Akt signaling in HPFs originating from A-CTGs implied their involvement in cell survival, proliferation, and motility. Prominent collagen-rich expression profile contributing to high mechanical stability, increased osteogenesis-related gene expression, and strongly activated (P < 0.001) Smad1/5/8 signaling characterized HPFs originating from P-CTGs. The present data indicate that in humans, differences between palatal CTGs harvested from different locations and depths appear to be location- rather than depth-dependent. Our findings provide the basis for future personalization of the therapeutic strategy by selecting an optimal graft type depending on the clinical indications.

Table S1: Primer sequences for genes belonging to the gene sets specifically upregulated in AD-versus PD-as well as in PS-versus PD-CTGs, and related to cell migration and immunomodulation.
Table S2: Primer sequences for genes belonging to the gene sets specifically upregulated in Aversus P-as well as in AD-versus PD-CTGs, and related to Erk and Akt signaling.
Table S3: Primer sequences for genes belonging to the gene sets specifically upregulated in Pversus A-as well as in PD-versus AD-CTGs, and related to ECM organization and connective tissue development.
Table S4: Primer sequences for genes belonging to the gene set specifically upregulated in Pversus A-CTGs and related to osteogenesis.Table S3: Primer sequences for genes belonging to the gene sets specifically upregulated in Pversus A-as well as in PD-versus AD-CTGs, and related to ECM organization and connective tissue development.Table S4: Primer sequences for genes belonging to the gene set specifically upregulated in Pversus A-CTGs and related to osteogenesis.

Figure S2 :
Figure S2: Cell viability (a) and proliferation (b) of primary AD-, AS-, PD-, and PS-HPF cells originating from different CTG types.

Figure S4 :
Figure S4: Increased expression of a number of chemokines validate the pro-migratory and immunomodulatory phenotype of AD-and PS-CTGs.

Figure S5 :
Figure S5: Increased growth factor gene expression in A-CTGs determine their role in cell survival, proliferation, and motility.

Figure
Figure S8 and S9: Uncropped immunoblots for various proteins investigated in the study.

Figure S1 .
Figure S1.Gene ontology overrepresentation analysis of the 208 transcript-set upregulated in AS-versus PS-CTGs (a), the 255 transcript-set upregulated in PS-versus AS-CTGs (b), the 33 transcript-set upregulated in AS-versus AD-CTGs (c), and the 65 transcript-set upregulated in PD-versus PS-CTGs (d) using the clusterProfiler.The high-level associations with biological processes (BP), molecular functions (MF), and cellular components (CC) are displayed along the x-axis of each bar chart.The y-axis displays the -log10 of the adjusted p-value.The blue vertical line denotes the cutoff for significance (p = 0.05).Broader functional categories combining related gene ontology terms are devised and color-coded in the legends to facilitate data interpretation.

Figure S2 .
Figure S2.No significant differences in cell viability (a) and proliferation (b) of primary AD-, AS-, PD-, and PS-HPF cells originating from different CTG types.Primary HPFs of each type were subjected to the CellTiter-Blue cell viability assay (a) and the BrdU Cell Proliferation ELISA (b) according to the manufacturers' protocols and as described in the Materials and Methods section in the main text.Data represent means ± SD from three independent experiments performed with three different HPF cell donors.

Figure S3 .
Figure S3.Pro-migratory effects of primary HPFs originating from AD-, AS-, PD-, and PS-CTGs.Migration of hTERT TIGK (a) and primary HEC (b) toward primary AD-, AS-, PD-, and PS-HPFs.For clarity, the abbreviation HPF is omitted and only the two letter-abbreviation (AD, AS, PD, and PS), indicating the origin of the HPF cell line from the respective CTG type is used.Bar charts present quantification of cell migration in the absence (Ctrl) or presence of HPFs by measuring the area on the lower side of the filter covered with migrated epithelial cells.Representative images of fixed and stained cells that have migrated to the lower side of the filter in each of the experimental groups are shown (a, b).Scale bar, 500 μm.Data represent means ± SD from three independent experiments performed with three different cell donors, in duplicates.Significant differences to the control unless otherwise indicated, ***p < 0.001, **p < 0.01.

Figure S4 .
Figure S4.Increased expression of a number of chemokines validate the pro-migratory and immunomodulatory phenotype of AD-and PS-CTGs.qRT-PCR analyses of CCL2, CCL8, CCL24, CCL26 (a), and CXCL10, CXCL11, CXCL13, and GZMB (b) transcripts normalized to GAPDH in AD-, AS-, PD-, and PS-CTG tissue samples.For clarity, the abbreviation CTG is omitted and only the two letter-abbreviation (AD, AS, PD, and PS), indicating the type of the CTG, is used.The affiliation of each transcript to the respective gene set is indicated in parentheses after the gene symbol.Data represent means ± SD from 16 samples per CTG type.Significant differences between experimental groups, ***p < 0.001, **p < 0.01, *p < 0.05.

Figure S5 .
Figure S5.Increased growth factor gene expression in A-CTGs determine their role in cell survival, proliferation, and motility.qRT-PCR analyses of EGF, HBEGF, FGF1, FGF9, FGF10, FGF12, GPC3, CEACAM1, EFEMP1, LEP, MYOC, and NTRK3 transcripts normalized to GAPDH in AD-, AS-, PD-, and PS-CTG tissue samples.For clarity, the abbreviation CTG is omitted and only the two letter-abbreviation (AD, AS, PD, and PS), indicating the type of the CTG, is used.The affiliation of each transcript to the respective gene set is indicated in parentheses after the gene symbol.Data represent means ± SD from 16 samples per CTG type.Significant differences between experimental groups, ***p < 0.001, **p < 0.01, *p < 0.05.

Figure S6 .
Figure S6.ECM-rich expression profile characterizes P-CTGs.qRT-PCR analyses of COL1A1, COL1A2, COL5A1, COL5A2, COL6A2, COL10A1, COL11A1, COL23A1, ADAMTS2, ADAMTS3, PCOLCE, and LOX transcripts normalized to GAPDH in AD-, AS-, PD-, and PS-CTG tissue samples.For clarity, the abbreviation CTG is omitted and only the two letter-abbreviation (AD, AS, PD, and PS), indicating the type of the CTG, is used.The affiliation of each transcript to the respective gene set is indicated in parentheses after the gene symbol.Data represent means ± SD from 16 samples per CTG type.Significant differences between experimental groups, ***p < 0.001, **p < 0.01, *p < 0.05.

Figure S7 .
Figure S7.Increased osteogenesis-related gene expression in P-CTGs.qRT-PCR analyses of RUNX2, DLX1, GATA3, CCN4, RSPO2, VCAN, SPP2, SPARC, and ASPN transcripts normalized to GAPDH in AD-, AS-, PD-, and PS-CTG tissue samples.For clarity, the abbreviation CTG is omitted and only the two letter-abbreviation (AD, AS, PD, and PS), indicating the type of the CTG, is used.The affiliation of each transcript to the respective gene set is indicated in parentheses after the gene symbol.Data represent means ± SD from 16 samples per CTG type.Significant differences between experimental groups, **p < 0.01, *p < 0.05.

Figure S8 .
Figure S8.Immunoblot analyses of phospho-p38 (pp38) (a), phospho-Erk1/2 (pErk1/2) (b), and phospho-Akt (pAkt) (c) proteins in whole-cell extracts from AD-, AS-, PD, and PS-HPF cells.Each of the blots was cut at the level of ~80 kDa.The upper part of each blot was developed for the vinculin loading control.The lower part of each blot was first developed for the respective phosphorylated protein.After stripping in buffer containing 62.5 mM Tris-HCl pH 6.8, 100 mM 2mercaptoetanol, and 2% SDS for 30 min at 50 0 C, the lower parts were subsequently blocked in 5% w/v BSA, 1x TBS, 0.1% Tween-20, and developed for the respective total proteins used as internal controls.M = protein marker.

Figure S9 .
Figure S9.Immunoblot analysis of collagen type I (a) and phospho-Smad1/5/8 (pSmad1/5/8) (b) proteins in whole-cell extracts from AD-, AS-, PD, and PS-HPF cells.Each of the blots was cut at the level of ~70-80 kDa.The upper part of the blot in (a) was developed for collagen type I protein whereas the lower part was developed for the GAPDH loading control.The upper part of the blot in (b) was developed for the vinculin loading control whereas the lower part was first developed for the pSmad1/5/8 protein.After stripping in buffer containing 62.5 mM Tris-HCl pH 6.8, 100 mM 2-mercaptoetanol, and 2% SDS for 30 min at 50 0 C, the lower part in (b) was subsequently blocked in 5% w/v BSA, 1x TBS, 0.1% Tween-20, and developed for the total Smad1 protein used as an internal control.M = protein marker.